SV40 encodes at least two transforming proteins: the 94K A protein (large T antigen) and the 17K F protein (small T antigen). The A protein regulates the initiation of viral DNA replication and transcription and is required to maintain all aspects of the transformed phenotype in some cell types. The F protein has no known function in productive infection and is required for the expression of a defined subset of the transformed phenotype including anchorage-independent growth and cytoskeletal structure. OBJECTIVES: We plan to study the structure and function of the A and F proteins in vitro and to detect structural or enzymatic interactions between the viral transforming proteins and specific cellular proteins. METHODS: Wild-type and mutant transforming proteins will be purified and modified by phosphatase or protease treatment. Possible interactions between viral and cellular proteins will be studied using gel filtration and cross-linking techniques. Findings will be compared with an analysis of protein interactions in nucleoprotein complexes assembled in vivo. Transient alterations in the structure of viral DNA after binding with the A protein will be investigated. The search for enzymatic activities of the A protein or associated cellular proteins will focus on possible protein kinase and RNA polymerase functions. If the A protein has kinase activity, cellular acceptor proteins will be characterized. Studies of the F protein function will focus on possible protease activity and on the disassemby of a triton-extracted cytoskeleton.